Enzymatic amplification and sequence analysis of precore/core DNA in HBsAg-positive patients

Serum or plasma from 69 HBsAg-positive patients was tested for the presence of precore core gene specific DNA by the polymerase chain reaction (PCR). In both healthy individuals (n = 26) and chronic carriers (n = 25), there was a strong correlation between presence of circulating anti-HBe and the absence of detectable HBV genome in serum. In 18 serum samples where HBsAg was the only detectable marker, i.e., anti-HBc-negative specimens, HBV DNA could be detected in three samples. HBV strains from 21 of the 24 PCR-positive samples were sequenced over the precore/core junction. A stop codon at the end of the precore region, described by other workers, was found in strains from two blood donors, one of whom had detectable HBeAg in serum. Conversely, HBV strains from the three anti-HBc-negative patients where DNA of the HBV precore region could be amplified and who had no detectable serum HBeAg or anti-HBe did not have this stop codon. The study indicates that further investigations are required before lack of HBeAg can be correlated with evidence of mutations in the precore region.     

Prevalence of precore mutants in anti-HBe-positive hepatitis B virus carriers in Germany.

Hepatitis B virus (HBV) precore mutants are associated often with highly productive infection in hepatitis B surface antigen (HBsAg) carriers lacking hepatitis B e antigen (HBeAg) but positive for anti-HBe, rendering serological identification of infectious individuals unreliable. Although considered initially to be limited mostly to the Mediterranean area, more recent studies suggest a significant presence of these mutants in northern European countries. The sequence of the precore region was determined and examined for mutations from HBV isolates of 99 German chronic HBsAg carriers positive for HBV-DNA and either HBeAg (n = 15) or anti-HBe (n = 84). In addition, clinical data of individuals carrying wild-type virus and those with precore mutants were compared. HBV precore mutants were found in more than half (44/84) of all HBeAg-negative, anti-HBe-positive virus carriers. There was no difference between carriers of wild-type and precore mutant HBV in the level of viremia or in the clinical course of chronic infection. In conclusion, HBV precore mutants are common in Germany and can therefore present a diagnostic problem for serological testing. However, precore mutants do not appear to have a detrimental effect on the course of chronic HBV infection.     

Presence of hepatitis B virus core promoter mutations pre-seroconversion predict persistent viral replication after HBeAg loss.

BACKGROUND: In patients with chronic hepatitis B virus (HBV) infection DNA levels do not always fall after anti-hepatitis B e (anti-HBe) seroconversion. OBJECTIVES: To follow longitudinally through HB e antigen (HBeAg) loss HBV DNA levels and core promoter/precore sequences in a cohort of 21 chronic HBV carriers. STUDY DESIGN: Treatment-na?ve HBeAg seropositive HBV carriers were monitored through HBeAg loss for between 2 and 22 years (mean 9.3). Core promoter/precore sequences, genotypes, HBV DNA levels and HBe status were determined. RESULTS: Patients were grouped into those in whom serum/plasma HBV DNA remained high after HBeAg loss (group 1, n=11; HBV DNA>5log(10)IU/ml) and those in whom HBV DNA declined (group 2, n=10). Re-appearance of HBeAg was seen in seven group 1 patients. Pre-seroconversion mutations in the core promoter region including A1762T and/or G1764A were detected more frequently in group 1 (P=0.031). Overall sequence changes at sites other than 1762/1764 were more common post-seroconversion in group 1 than group 2 patients (P=0.037). CONCLUSIONS: The presence of core promoter mutations prior to HBeAg loss identified those patients in whom HBV DNA persisted at high levels and was associated with temporary re-emergence of serum HBeAg. These patients may benefit from early anti-viral treatment.     

乙丙型肝炎病毒重叠感染时病毒的相互作用

目的　探讨乙、丙型肝炎病毒（ＨＢＶ、ＨＣＶ） 重叠感染时,病毒之间的相互作用。方法　检测３０ 例ＨＢＶ、ＨＣＶ重叠感染患者血清病毒标志物的变化、ＨＢＶ前Ｃ区１ ８９６ 位点突变的发生比率及血清肿瘤坏死因子（ＴＮＦ）α、白细胞介素（ＩＬ）６ 含量。结果　与单纯ＨＢＶ或ＨＣＶ 感染者相比,重叠感染患者乙肝表面抗原（ＨＢｅＡｇ） 、ＨＢＶＤＮＡ、ＨＣＶＲＮＡ 阳性比率明显降低,乙肝ｅ 抗体（ 抗ＨＢｅ） 阳性比率明显升高,ＨＢｓＡｇ、抗ＨＢｃ ＩｇＧ 及抗ＨＣＶ 几何平均滴度也明显降低,部分患者ＨＢｓＡｇ 阴转。而ＨＢＶ前Ｃ区１ ８９６ 位点突变发生率及血清ＴＮＦα、ＩＬ６ 含量却明显高于单纯感染者。结论　ＨＢＶ、ＨＣＶ感染同一宿主时,存在相互干扰、抑制;ＨＢｅＡｇ 的消失、抗ＨＢｅ 的阳转既与ＨＣＶ对ＨＢＶ 复制的直接抑制有关,又与ＨＢＶ的前Ｃ区变异有关,且ＨＣＶ 的重叠感染可能是导致ＨＢＶ 前Ｃ区变异的原因之一,其作用机制可能与导致机体免疫压力升高有关     

Longitudinal study of hepatitis activity and viral replication before and after HBeAg seroconversion in chronic hepatitis B patients infected with genotypes B and C

The aims of this study were to compare chronic hepatitis B (CHB) patients with genotypes B and C for the probability of HBeAg seroconversion, hepatitis activity, and viral replication before and after HBeAg seroconversion and to compare the prevalence of core promoter and precore mutations. A total of 180 asymptomatic Chinese patients with CHB were monitored for a median of 53.8 months, and 38 patients with cirrhosis-related complications were studied. Hepatitis B virus (HBV) DNA levels were measured in 16 patients with HBeAg seroconversion at 3 months before, during, and 3 months after HBeAg seroconversion and in all patients at the last follow-up. Hepatitis B genotypes and core promoter and precore mutations were determined. Compared to patients with genotype C (n = 109), patients with subtype Ba (n = 69) had a higher rate of anti-HBe positivity on presentation (P = 0.05). HBeAg-positive patients with subtype Ba had a higher cumulative rate of HBeAg seroconversion than patients with genotype C (P = 0.02). However, there were no differences between the two groups with regard to the median HBV DNA levels before, during, and after HBeAg seroconversion; the probability of having persistently normal or elevated aminotransferase levels; and the median HBV DNA levels and liver biochemistry at the last follow-up. There was no difference in the prevalence of genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications. Though patients with subtype Ba had earlier HBeAg seroconversion, the liver biochemistry, HBV DNA levels in different phases of the disease, and the probability of development of cirrhosis-related complications were the same with genotypes Ba and C.     

Hepatitis B virus DNA in asymptomatic HBsAg carriers: comparison with HBeAg/anti-HBe status

Sera of 17 HBeAg positive and 104 anti-HBe positive asymptomatic HBsAg carriers from two cohorts were tested for HBV DNA. HBV DNA was found in 13 of 17 HBeAg positive carriers (76.5%) and in only 7 of 104 of anti-HBe positive carriers (6.7%). Eleven of the 17 HBeAg positive carriers were retested for HBV DNA over a period of 7 to 36 months after the initial test. HBV DNA disappeared from the serum in 2 patients in spite of persistence of the HBe antigen. Of the 104 anti-HBe carriers, 89 were retested for HBV DNA over a period of 6 to 52 months after the initial test. HBV DNA disappeared from the serum in 5 of the 7 who were previously positive for HBV DNA, and persisted in 2. These findings indicate that there is an inconstant relationship between the time of seroconversion of HBeAg to anti-HBe and the disappearance of HBV DNA. In one HBeAg positive patient, HBV DNA, which was absent in the serum on first testing, was present on retesting. This suggests that the presence of HBV DNA in the serum of some patients may be intermittent. The presence of HBV DNA in the serum of some anti-HBe positive carriers accounts for the finding that they may be infective. All but one of the HBV DNA positive anti-HBe carriers were born outside North America, most in Asia. HBV DNA were found more frequently in the serum of anti-HBe positive carriers who had biochemical and histological evidence of liver disease than in carriers without such evidence.     

High frequency of hepatitis B virus DNA in anti-HBe positive sera on longitudinal follow-up of patients with renal transplants and chronic hepatitis B

Hepatitis B virus (HBV) markers were determined in 821 patients receiving renal allografts and undergoing immunosuppressive therapy during 1970-1986. Twenty-four of the patients with a renal transplant functioning for longer than 1 year originally were or became chronic carriers of hepatitis B surface antigen (HBsAg). These patients remained carriers during the follow-up period, which lasted until death or until the end of 1986. Follow-up time was 1.2-15.3 years (mean 9.1 years). A total of 301 samples from the HBsAg-positive patients were tested for HBV DNA and HBeAg/DNA and HBeAg/anti-HBe. Nine patients who were constantly positive for HBeAg also remained positive for HBV DNA. Reactivation of HBV replication occurred in 11 patients. Among these, HBV DNA and HBeAg varied in parallel in six patients, three patients developed anti-HBe, and two patients were constantly positive for anti-HBe. Another four of the 24 patients seroconverted to anti-HBe, and two of these also lost HBV DNA. Three of 12 deceased patients died from liver failure during follow-up. None of these three had been constantly positive for HBeAg or HBV DNA, but they had had reactivations of HBV; two were also positive for HBV DNA in serum specimens available from their terminal month. HBV DNA was demonstrated in 99% of HBeAg-positive and 53% of anti-HBe-positive sera and in at least two samples from each of the 24 patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidemiology of precore mutants of hepatitis B in the United Kingdom

A point mutation assay was used to study the codon 28 and codon 1 precore mutant status of 310 chronic hepatitis B carriers (82 HBeAg positive and 228 HBeAg negative). Fourteen of 228 (6%) of HBeAg negative carriers had high levels of serum HBV DNA. Nine of these were explained by precore variants, three by core promoter variants, and two were not explained by recognised precore changes. Nested PCR detected serum HBV DNA in 36% (82/228) of HBeAg negative carriers and 63% (52/82) of these had precore variants. Four of 82 (4%) of the HBeAg positive carriers had precore variants, all as mixed mutant/wild type populations and evidence indicated that these carriers were seroconverting. Overall 23% (52/228) of HBeAg negative carriers had both serum HBV DNA and codon 1 or 28 precore mutations. A sexual transmission event from an HBeAg negative carrier with a relatively low serum HBV DNA level (10(4)-10(6) genome copies/ml) and only core promoter mutations was observed. Despite high rates of variant carriage in the antenatal sub-group perinatal transmission was not observed. The results of direct sequencing on 45 carriers validated the point mutation assay and also showed that codon 28 mutations were only seen in carriers with the genotype CCT at codon 15. For the Caucasian population a higher prevalence of codon 28 mutations (13/25 or 52%) than expected was seen. Liver biopsy data indicated that there was no link between the presence or absence of precore mutants and the severity of liver disease.     

Anti-HBc titers in HBeAg and anti-HBe positive asymptomatic HBsAg carriers

A study was undertaken to establish markers for HBV replication in relation to HBeAg and anti-HBe. HBsAg carriers with serum HBeAg had DNA polymerase activity in the serum and HBcAg in the liver nuclei. Anti-HBe positive and anti-HBe/HBeAg negative sera lacked these markers. For anti-HBc the following geometrical mean titers were calculated: 1: 12,000 for HBeAg positive, 1:9, 100 for anti-HBe and anti-HBc positive, and 1:2,800 for anti-HBc positive anti-HBe/HBeAg negative asymptomatic HBsAg carriers. Follow up studies revealed mostly unchanged anti-HBc titers in all three groups over an observation period of ten to twenty months. Our data argue for a prolonged HBV replication in all HBsAg carrier subgroups compared to individuals with an uncomplicated acute virus-B-hepatitis. This study gives no final answer whether HBeAg negative HBsAg carriers have a continous HBV replication.     

Conservation of precore and core sequences of hepatitis B virus in chronic viral carriers

Mutations in the precore region of hepatitis B virus (HBV) have been associated with failure of expression of HBV e-antigen (HBeAg), however, the prevalence of these and other mutations in HBV carriers without overt chronic liver disease remains uncertain. Homosexual or bisexual males (n = 65) with chronic HBV infection attending The Middlesex Hospital, London were studied, of whom two had clinical evidence of chronic liver disease. HBV DMA was amplified from 62 of 65 serum samples using nested and double nested polymerase chain reaction (PCR) assays. Direct sequencing of the PCR products was employed to investigate sequence variation. HBV-DNA from all available HBeAg-negative (n = 9) and selected HBeAg-positive (n = 33) sera were sequenced in the entire precore gene, the 3′ terminal portion of the X gene (aa128–154), and the 5′ terminus of the core gene (aa18–73). Sequences were highly conserved in all regions studied. Samples from two anti-HBe-seropositive patients contained mutations in the precore region. In one, a single mutation in the first amino acid resulted in a change to leucine, which would prevent translation of this region and therefore HBeAg expression. Wild type sequences were also detected in this sample. In the other sample from a patient with overt chronic liver disease, a mutation of precore amino acid 28 changed a tryptophan residue to a stop codon which would also prevent HBeAg expression. Although few such patients were studied, precore mutations may be uncommon in anti-HBe-seropositive patients without overt chronic liver disease. Possibly such mutations are not related to HBeAg to anti-HBe seroconversion, but rather they may arise in patients who remain anti-HBe seropositive for prolonged periods and they may be causally associated with the development of chronic liver disease. © 1994 Wiley-Liss, Inc.     

HBV DNA levels and transmission of hepatitis B by health care workers.

BACKGROUND: Laboratory-based study funded by the Research and Development Division of the Department of Health to inform the decision making on guidelines for the conduct of exposure prone procedures (EPPs) by health care workers who are hepatitis B carriers. OBJECTIVES: Define the quantity and nature of hepatitis B virus (HBV) DNA in hepatitis carriers whose serum does not contain hepatitis B e antigen (HBeAg) and in surgeons previously cleared to conduct EPPs who have transmitted HBV to their patients. STUDY DESIGN: Cross-sectional survey using HBV DNA quantification, genotyping and sequencing comparing transmitting surgeons and asymptomatic carriers. RESULTS: HBV DNA could be detected and quantified in 64.5% (136 of 211) of carriers whose serum did not contain HBeAg with a median level 3.6 log(10) copies/ml (range of 5.7 log(10) copies). Pre-core mutation appeared not to affect the HBV DNA level, however, all surgeons carried codon 28 variants and transmitted these variants to their patients. The lowest HBV DNA level in a transmitting surgeon was 4 x 10(4) copies/ml. CONCLUSIONS: Pre-core mutations are common in carriers whose serum does not contain HBeAg and do not specifically identify carriers whose HBV DNA levels are high. It was possible to define a level of virus above which transmission of hepatitis B during conduct of EPPs could not be excluded.     

Clinical and serological variation between patients infected with different Hepatitis B virus genotypes

Hepatitis B virus (HBV) has eight genotypes which have distinct geographical distributions. Studies comparing differences in the clinical outcomes of infections caused by strains with genotype-related variations in the HBV genome have largely compared genotypes B and C and genotypes A and D but not all four genotypes. The present study included 196 HBV-infected patients attending an infectious diseases outpatient clinic in Sweden. The age and geographic origin, liver function, HBeAg and anti-HBe status, and the presence or absence of HBV DNA were analyzed for each patient. HBV DNA was detected in 144 patients, and the HBV genotype and the core promoter and precore sequences were determined for the isolates from 101 of these patients. Among the patients who might be considered most likely to be nonviremic, namely, anti-HBe-positive HBV carriers with normal alanine aminotransferase (ALT) levels, 65% had detectable HBV DNA and were thus viremic. Among the viremic patients, HBeAg-positive patients were more likely to have elevated ALT levels than anti-HBe-positive patients. HBV genotypes A to F were represented in the study, and their distributions coincided accurately with the origin of the patient. A significantly higher number of genotype D-infected patients were anti-HBe positive and had elevated ALT levels (42% of genotype D-infected patients but 0% of patients infected with genotypes B and C). Genotype D strains with mutations in the core promoter and precore regions were significantly correlated with elevated ALT levels in the patients. The differences were not age related. Therefore, in this large-scale cross-sectional study, genotype D appears to be associated with more active disease.     

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.     

Serum hepatitis B virus DNA: relationship with the e-antigen and anti-e status in chronic carriers

The relationship between serum Hepatitis B virus DNA (HBV-DNA) and the Hepatitis B e-antigen/ anti-Hepatitis Be (HBeAg/anti-HBe) serological status in Malaysians was studied. 212 cases of asymptomatic HBV carriers were recruited for this study. 92 cases were positive for the HBeAg at the point of recruitment. 85 (92.4%) of these patients tested positive for HBV-DNA, of whom 55 (64.7%) had levels over 100pg/ml of serum. Three of the remaining 7 HBeAg positive cases who were negative for HBV-DNA subsequently seroconverted. The other 4 cases remained negative for HBV-DNA for periods of 6-12 months. Out of 113 cases who were anti-HBe positive, 12 (10.6%) gave a positive HBV-DNA result. 2 of these 12 patients were recent seroconverters; the remaining cases had transiently increased viral replicative activity which later subsided. 7 out of the 212 carriers were in the e-window period; all 7 tested negative for HBV-DNA. Our data confirm a high frequency of HBV-DNA in HBeAg positive carriers and a negative correlation between HBV-DNA and anti-HBe. An atypical profile of anti-HBe associated with HBV-DNA was observed in 10.6% of the carriers. An inverse relationship between serum HBV-DNA levels and age was also observed.     

Reactivation of precore variant hepatitis B virus in a child with severe aplastic anaemia

Reactivation of hepatitis B e antigen (HBeAg) negative chronic hepatitis B virus (HBV) infection due to selection of precore variant virus is an uncommon complication of previous hepatitis B infection, and virtually unrecognised in children and adolescents. A child who had received treatment with methylprednisolone and antilymphocyte globulin for severe aplastic anaemia developed high levels of detectable HBV DNA associated with hepatitis B e antibody (anti-HBe) positivity. HBV DNA was extracted, amplified and the core and precore regions sequenced from 2 samples. A mixture of wild-type and the precore variants A(1896) and A(1899) was detected in both samples, with the wild-type predominating in the second sample. Reinfection was excluded by phylogenetic analysis using Phylip and the neighbour-joining method. Precore variant Hepatitis B virus can be transmitted to children as a primary infection, and it is important that aggressive liver disease, particularly in the presence of the anti-HBe phenotype, be investigated. Further studies are needed to determine the frequency of these variants.     

A case-control study for early prediction of hepatitis B e antigen seroconversion by hepatitis B virus DNA levels and mutations in the precore region and core promoter

Factors influencing and predictive of seroconversion from hepatitis B e antigen (HBeAg) to antibody (anti-HBe) were sought in a case-control study of 61 patients with chronic hepatitis B who had been observed from 5 years before to 1 year after seroconversion, and 32 patients who did not seroconvert during the entire 6-year period. Almost all of the patients (96%) were infected with HBV genotype C. HBV DNA levels began to decrease 3 years before seroconversion in the seroconverters, while they remained high in the non-converters. The frequency of precore mutation and the loss of HBeAg (A1896) started to increase 1 year before in the converters, and became significantly higher at seroconversion (23 vs. 3%, P = 0.030) than that in the non-converters. Double mutation in the core promoter (T1762/A1764) was more common in the seroconverters than in the non-converters 5 years before seroconversion (48 vs. 28%), and became significantly more frequent at seroconversion (65 vs. 41%, P = 0.046). Seroconversion occurred in 75% of the patients with at least HBV DNA levels <5.5 logarithmic equivalents/mL; precore mutation in 20% or more of HBV DNA; or core promoter mutation. Seroconversion occurred in 50% of those patients within 1 year, 88% within 2 years, and 93% within 5 years. These results indicate that a decrease in HBV DNA levels and mutations in the precore region and the core promoter were associated significantly and complementarily with seroconversion, and each of them or a combination thereof was predictive of seroconversion years ahead.     

Diagnosing different stages of hepatitis B infection using a competitive polymerase chain reaction assay

PURPOSE: Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. METHODS: One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. RESULTS: The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 x 10(8) vs. 2.30 x 10(5) copies/mL ; P<0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 x 10(5) vs. 4.28 x 10(3) copies/mL; P<0.05). The 2.5 x 10(4) copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. CONCLUSIONS: Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 x 10(4) copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease.     

Clinical and Virological Aspects of Blood Donors Infected with Hepatitis B Virus Genotypes B and C

Pathogenic and therapeutic differences among hepatitis B virus (HBV) genotypes have been documented. However, the association of virological characteristics with clinical differences among HBV genotypes remains unclear. We therefore studied the clinical and virological characteristics of Taiwanese volunteer blood donors infected with HBV genotypes B and C. HBV genotypes were determined in 300 candidate blood donors positive for HBV surface antigen (HBsAg), and sequences of the precore gene of the HBV genome were determined in 50 HBV e antigen (HBeAg)-positive and 50 HBeAg-negative blood donors. Of 300 HBsAg-positive blood donors, 10% had elevated serum aminotransferase levels and 27% were positive for HBeAg. HBV genotype distribution in 264 viremic carriers was as follows: B, 221 (83.7%); C, 39 (14.8%); F, 1 (0.4%); and mixed infection, 3 (1.1%). Blood donors with genotype C infection tended to have a higher frequency of HBeAg positivity and a higher serum HBV DNA level than those with genotype B infection. The frequency of precore stop codon mutation was significantly higher in HBeAg-negative blood donors than HBeAg-positive ones, irrespective of HBV genotypes. Meanwhile, only 5% of blood donors with genotype C infection had C-1858 strains. In conclusion, mixed infection of HBV genotypes indeed occurs, and genotype C has a higher serum HBV DNA level than genotype B. Precore stop codon mutation is common in HBeAg-negative HBV carriers, irrespective of HBV genotypes. In contrast, precore C-1858 strains are rarely identified in Taiwanese HBV genotype C.